Document Type : Original Research Papers
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic or
mitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from the
intermembrane space of mitochondria and binding to Apaf-1 molecules, a heptameric
apoptosome complex is formed and triggers the downstream cascade of caspases. Here, for the
first time we present spontaneous mutations and recombinations of the Apaf-1 gene and its
neighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector with
Nluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesis
method, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37ºC. In 21 of
these cases, we found 38 different mutations. Our data suggest that there is a direct relationship
between bacterial incubation temperatures and the number of unwanted spontaneous mutations.
During our experiment we found that the Apaf-1 gene is much less susceptible to spontaneous
mutations when it is transformed into XL10-Gold at 18 ºC . In contrast, a large number of
spontaneous mutations were found when the gene of interest transformed into DH5α at 37ºC .